| Camellia oleifera,belongs to genus Camellia,is a unique woody oil crop in China.Theasaponin is one of the main active components in C.oleifera,which has many biological activities such as bacteriostasis,anti-inflammatory and anti-tumor.It is difficult to separate and purify theasaponin monomer,and new saponin species are rarely reported,and its chemistry and biological function are largely untapped.In this study,the chemical components of different organs of C.oleifera,as well as C.oleifera seed cakes were studied.Base on the analysis,we performed a systematic extraction,purification,and structural identification of teasaponin in C.oleifera seed cake.Moreover,the anti-inflammatory and antitumor activities of the isolated teasaponin were investigated.The main research results are showed as follows:1.There are certain differences in saponin types and contents among different organs of C.oleifera.The leaves,shell,seed coat,kernel and other organs of ’Huashuo’were collected respectively,and the C.oleifera seed cake was also used for further analysis.A total of 67 teasaponins,along with 24 unknown ones,were identified in the tested samples according to their mass spectrometric behavior.Teasaponin was found in different organs of C.oleifera,with variations in types and contents.The contents of saponin in different organs of C.oleifolia were ranked as follows:seed kernel/shell>seed coat>leaf;The types of saponin in different organs of C.oleifolia were ranked as follows:seed kernel>seed coat>shell/leaf.Camelliasaponin B1/B2、Camelliasaponin A1/A2、oleiferasaponin A1 were all detected in fruit shell,seed coat,seed kernel and C.oleifolia seed cake.2.C.oleifera seed cake is rich in saponin resources.The variety and content of saponin in the C.oleifera seed cake were similar to those in the seed kernel,which exhibited both remarkable variety and high amount of saponin among all tested C.oleifera samples.C.oleifera seed cake contained 43 kinds of teasaponins,along with 16 unknown ones;The yield of crude saponin extract of C.oleifera seed cake was 5.32%.In addition,the metabonomics study of C.oleifera seed cake showed that the main component was flavonoids,and the relative content of theasaponin was 6.34%,which contained 19 possible new theasaponin components.C.oleifera seed cake has high saponin extraction efficiency,rich saponin resources and many new thesaponin structures.It is an excellent raw material for saponin extraction and has great development and utilization value.3.In searching for novel natural products,the extracts of C.oleifera seed cake were chemically examined by using various chromatographic technologies and their structures were confirmed via extensive use of NMR spectroscopy as well as HR-MS analysis.As a result,four teasaponins were isolated and identified,including two new ones.The known teasaponins are identified as camelliagenin A 16-tiglate(2)and camelliagenin A 22-tiglate(3)by comparing their spectral data with reported literature.Structurally,the new teasaponin 1 is an aldehyde product of known compound camelliagenin A 16-tiglate(2)at C23 position.The molecular formula of the new teasaponin 4 is C47H71O14,and it is also an aldehyde derivative of teasaponins.Compared to camelliagenin A 22-tiglate(3),the teasaponin 4 were additionally substituted by aldehyde group at C23,tiglic acid group at C21,and a monosaccharide group at C3,respectively.4.In order to develop promising drug leads from teasaponins,the antibacterial,anti-inflammatory,PCSK9 inhibitory and anti-tumor activities of the isolated teasaponins were evaluated.The results of antibacterial test showed that the four teasaponins had no obvious antibacterial effects on 10 strains of entomopathogenic fungi.The results of anti-inflammatory effects showed that camelliagenin A 22-tiglate(3)exhibited considerable NO inhibitory activity.In vitro PCSK9 inhibitor assay showed that the new compound 4 had potent PCSK9 inhibitory activity with an IC50 value of 80.6μM.The results of anti-tumor experiment showed that all four compounds represented remarkedly anti-tumor activity.Compound 3 showed selective antitumor activity against 8 tumor cell lines,with the strongest cytotoxicity against human glioma cell line U251(IC50 of 2.4 μm)and the weakest cytotoxicity against human hepatocellular carcinoma cell line Huh-7(IC50 of 101.0 μm).Moreover,the antitumor activity(IC50 ranged from 1.5 μM to 11.3 μm)of 4 was stronger than that of the positive control cisplatin.Preliminary mode-of-action studies of compounds 3 and 4 showed that teasaponins could induce apoptosis of tumor cells by down-regulating p-mTOR,pSTAT3 and Bcl-2 levels.Compound 4 may trigger apoptosis through the Bcl2/Caspase-3 and JAK2/STAT3 pathways,and stimulate cell proliferation via the NFκB/iNOS/COX-2 pathway.Moreover,it was surprising to find that Compound 4 can inhibit the Nrf2/HO-1 pathway,which indicates that Compound 4 has the potency to overcome the resistance to cancer drugs.Therefore,Compound 4 are newly identified theasaponin derivatives with antitumor activity from the C.oleifera seed cake,and Compound 4 is a promising antitumor candidate not only for its powerful antitumor activity,but also for its ability to function as an Nrf2 inhibitor to enhance the anticancer drug sensitivity.In summary,the research results of this project provide both important theoretical basis and valuable material basis for developing bioactive small molecules from C.oleifera seed cake,which is the by-product of Camellia oil.The finding of the present study provided a foundation for realizing secondary development and utilization of agricultural supplementary products of C.oleifera seed cake,and accelerating the health industry promotion within the entire industrial chain of camellia oil. |
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